Methods of producing T memory stem cell populations

ABSTRACT

Provided are methods of producing an isolated T memory stem cell population, the method comprising a) isolating naïve T cells from a mammal, wherein the mammal is not a mouse; b) activating the naïve T cells and expanding the numbers of naïve T cells in the presence of one or more non-specific T cell stimuli, one or more cytokines, and a GSK-3beta inhibitor. Also provided are methods of producing an isolated T memory stem cell population, the method comprising a) isolating lymphocytes from a mammal; b) sorting the lymphocytes using flow cytometry into a population comprising a phenotype comprising i) CD95+, CD45RO−, and CCR7+; and ii) CD62L+ or one or more of CD27+, CD28+, CD45RA+, and CD127+ to produce an isolated T memory stem cell population. Further embodiments of the invention provide related cells, populations of cells, pharmaceutical compositions, and methods of treating or preventing cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 14/425,713, filed Mar. 4, 2015, which issued asU.S. Pat. No. 10,316,289 on Jun. 11, 2019, which is a U.S. NationalPhase of International Patent Application No. PCT/US2012/053947, filedSep. 6, 2012, which are incorporated by reference in their entiretiesherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberZ01ZIABC010763 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using tumor reactive T cells can producepositive clinical responses in cancer patients. Nevertheless, severalobstacles to the successful use of ACT for the treatment of cancer andother diseases remain. For example, T cells isolated from the peripheralblood of a host may not exhibit sufficient tumor-specific reactivity orpersist in the peripheral blood upon reinfusion into patients.Accordingly, there is a need for improved methods of obtaining apopulation of antigen-specific T cells from the peripheral blood of ahost that exhibit sufficient tumor-specific reactivity and which persistin the blood of patients.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of producing anisolated T memory stem cell population, the method comprising (a)isolating naïve T cells from a mammal, wherein the mammal is not amouse; and (b) activating the naïve T cells and expanding the numbers ofnaïve T cells in the presence of one or more non-specific T cellstimuli, one or more cytokines, and a glycogen synthase kinase(GSK)-3beta inhibitor.

Another embodiment of the invention provides a method of producing anisolated T memory stem cell population, the method comprising (a)isolating lymphocytes from a mammal; and (b) sorting the lymphocytesusing flow cytometry into a population comprising a phenotype comprising(i) CD95+, CD45RO−, and CCR7+; and (ii) CD62L+ or one or more of CD27+,CD28+, CD45RA+, and CD127+ to produce an isolated T memory stem cellpopulation.

Still another embodiment of the invention provides a method of producingan isolated T memory stem cell population, the method comprising (a)isolating lymphocytes from a mammal; and (b) sorting the lymphocytesusing flow cytometry into a population comprising a phenotype comprising(i) CD95+ and/or CXCR3+; and (ii) CD45RA+, CCR7+, and CD28+ to producean isolated T memory stem cell population.

Another embodiment of the invention provides an isolated or purified Tmemory stem cell comprising a phenotype comprising: (a) CD95+, CD45RO−,and CCR7+; and (b) CD62L+ or one or more of CD27+, CD28+, CD45RA+, andCD127+.

Yet another embodiment of the invention provides an isolated or purifiedT memory stem cell comprising a phenotype comprising (a) CD95+ and/orCXCR3+; and (b) CD45RA+, CCR7+, and CD28+.

Additional embodiments of the invention provide related populations ofcells, pharmaceutical compositions, and methods of treating orpreventing cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B are graphs showing the percentages of circulating CD8⁺(1A) and CD4⁺ (1B) naïve T cells (T_(N)), memory stem cells (T_(SCM)),central memory T cells (T_(CM)), or effector memory T cells (T_(EM)) in29 healthy donors.

FIGS. 1C and 1D are graphs showing expression of RORC (1C) and IL17A(1D) relative to ACTB by mucosal-associated invariant T (MATT) cells,T_(N), T_(SCM), T_(CM), or T_(EM) cells as measured by quantitativereverse-transcriptase polymerase chain reaction (RT-PCR).

FIG. 2A is a graph showing TREC copy number in sorted CD8⁺ T_(N),T_(SCM), T_(CM), or T_(EM) relative to T_(N) cells. Data are representedas means±standard error of the mean (s.e.m.) of four donors.

FIGS. 2B-2D are graphs showing the percentages of CD8⁺ T_(N), T_(SCM),T_(CM), or T_(EM) (from 6 healthy donors) producing interferon (IFN)-γ(2B), interleukin (IL)-2 (2C) or tumor necrosis factor (TNF)-α (2D) 4hours after exposure to Staphylococcus enterotoxin B.

FIGS. 3A-3C are graphs showing the percentages of CD8⁺ T_(N), T_(SCM),T_(CM), or T_(EM) (from 6 healthy donors) producing IFN-γ (3A), IL-2(3B) or TNF-α (3C) 4 hours after stimulation with α-CD3/CD2/CD28 beads.

FIGS. 4A-4C are graphs showing IFN-γ (4A), IL-2 (4B) or TNF-α (4C)release by sorted CD8⁺ T_(N), T_(SCM), T_(CM), or T_(EM) after 24 hourstimulation with CD3/CD2/CD28 beads.

FIGS. 5A and 5B are graphs showing the percentage of divided cells (5A)and proliferation index (5B) of different CD8⁺ T_(N), T_(SCM), T_(CM),or T_(EM), after stimulation with 25 ng ml⁻¹ of IL-15 for 10 days. Dataare represented as means±s.e.m. of 9 donors.

FIG. 5C is a graph showing the percentage of tetramer-binding cellsexpressing CD95 in the NL (CD45RO⁻CCR7⁺CD45RA⁺CD27⁺IL7Rα⁺) gate,determined by flow cytometry. Data represent the donors tested fortetramer specificity. HD, healthy donor; MP, melanoma patient.

FIGS. 6A-6L are graphs showing the robust multichip analysis(RMA)-normalized intensity of selected genes progressively downregulated(naïve associated genes LEF1 (6A), ACTN1 (6B), FOXP1 (6C), IL6ST (6D),LASS6 (6E), or TAF4B (6F)) or upregulated (effector associated genesEOMES (6G), GZMA (6H), TBX21 (6I), PRF1 (6J), PRDM1 (6K), or KLRG1 (6L))from T_(N) cells, T_(SCM) cells, T_(CM) cells, or T_(EM) cells. Data arerepresented as means±s.e.m. of three donors.

FIGS. 7A and 7B are graphs showing the percentage of CD8⁺ T cellsexpressing CCR7 and CD62L (7B) and CD45RA (7A) relative to cell divisionafter exposure to 25 ng ml⁻¹ of IL-15 for 10 days. Slopes were comparedusing a Wilcoxon rank test, *P=0.0391. The phenotype of sorted CD8⁺ Tcell subsets before stimulation is indicated as “Pre.”

FIG. 7C is a graph showing the percentage of carboxyfluoresceindiacetate succinimidyl ester (CFSE)-diluted CD8⁺ T_(SCM), T_(CM), orT_(EM) that retained the parental phenotype after stimulation with 25 ngml⁻¹ of IL-15 for 10 days. *P<0.05; **P<0.01 (t test).

FIG. 7D is a graph showing the self-renewal index (SI) of CD8+ memory Tcell subsets following secondary stimulation with 25 ng ml⁻¹ of IL-15.SI was calculated as follows: SI=2^(PI)P_(RP), PI=Proliferation Index,P_(RP)=Percent of cells retaining the input phenotype. Graph depicts theresults from 4 healthy donors; *p=<0.05.

FIGS. 8A and 8B are graphs showing the percentage of CD8⁺ T_(SCM)(circles) T_(CM) (triangles) or T_(EM) (diamonds) expressing CCR7 andCD62L (8B) and CD45RA (8A) relative to cell division after stimulationwith α-CD3/CD2/CD28-coated beads for 6 days. The phenotype of sortedCD8⁺ T cell subsets before stimulation is indicated as “Pre.”

FIG. 8C is a graph showing the sternness index of CD8⁺ T_(SCM), T_(CM),and T_(EM). Data are represented as means±s.e.m. of 4 donors. *P<0.05 (ttest).

FIG. 9A is a graph showing ³H-thymidine incorporation by sorted CD8⁺T_(N), T_(SCM), T_(CM), or T_(EM) after stimulation withα-CD3/CD2/CD28-coated beads. Data are represented as means±s.e.m. of tendonors. Results are normalized to the number of seeded cells, asdifferent cell numbers were obtained from different sorts. c.p.m.,counts per min. *P<0.05; **P<0.01; ***P<0.001 (t test).

FIGS. 9B-9G are graphs showing total human CD8⁺ T cell recovery in thespleens (9B), lymph node (LN) (9C) livers (9D), blood (9E), bone marrow(9F), or lungs (9G) from six NSG mice 4 weeks after adoptive transfer ofCD4⁺ T cells with or without sorted CD8⁺ T_(N), T_(SCM), T_(CM), orT_(EM). A total of six mice per T cell subset from two independentexperiments (three replicate mice per T cell subset per experiment) areshown. Horizontal bars indicate median values. *P<0.05; **P<0.01 (ttest).

FIGS. 10A and 10B are graphs showing percentage change of body weight(10A) or survival (10B) of untreated (*) mice or NSG mice bearingM108-luciferase mesothelioma after adoptive transfer of CD4⁺ T cells(10⁶) with or without (▾) sorted CD8⁺ T_(SCM) (circles), T_(CM) (▴), orT_(EM) (diamonds) (3×10⁶) expressing a mesothelin-specific chimericantigen receptor. ***P<0.001, one-way repeated measures ANOVA (e) andlog-rank (Mantel-Cox) test (f).

FIGS. 11A-11D are graphs showing the gating strategy for theidentification of human and rhesus T_(SCM) cells. Human and NHP PBMC arestained as indicated in Table 1, Panel #1 and Panel #3, respectively.Both panels include anti-CD4 conjugated to Qdot 585 in addition toanti-CD8 conjugated to Pacific Blue, to allow the simultaneousidentification of CD4+ and CD8+ T cells (11D). These T cells areidentified by first gating on singlets (FSC-H vs. FSC-A) (11A), liveCD3+ T cells (CD3 vs. Dump/AQUA) (11B) and lymphocytes (SSC vs. FSC)(11C).

FIGS. 11E-11L are graphs showing human cells sorted for CCR7 and CD45ROexpression (CD8+ in 11E and CD4+ in 11I); CD62L and SSC expression (CD8+in 11F and CD4+ in 11J); CD95 and CCR7 expression (CD8+ in 11G and CD4+11K); and CD95 and CCR7 expression gated on T cells (CD8+ in 11H andCD4+ 11L).

FIGS. 11M-11V are graphs showing rhesus cells sorted for CCR7 and CD45RAexpression (CD8+ in 11M and CD4+ in 11R); CD28 and CD95 expression (CD8+in 11N and CD4+ in 11S); CD95 and CXCR3 expression (CD8+ in 11O and CD4+11T); CD95 and CCR7 expression (CD8+ in 11P and CD4+ in 11U); and CD95and CCR7 expression gated on T cells (CD8+ in 11Q and CD4+ 11V). In NHPCD8+ T cells, CXCR3 is co-expressed with CD95 and thus helps to identifyCD8+ T_(SCM) cells, but not CD4+ T_(SCM) cells, as not all CD95+ T_(SCM)in naïve-like CD4+ cells express CXCR3+ (arrow; 11T).

FIGS. 11W-11Z are graphs showing the CD95 FMO control in human CD4+cells sorted for CD95FMO and CCR7 expression (11W); CD4+ cells sortedfor CD95Cy5PE and CCR7 expression (11X); CD8+ cells sorted for CD95FMOand CCR7 expression (11Y); and CD95Cy5PE and CCR7 expression. Dashed barindicates the threshold for positivity for CD95 expression while thediagonal bar indicates the T_(SCM) gate.

FIGS. 12A-12B are graphs showing the percentage (n=11) of CD8⁺ or CD4⁺naïve-like T cells identified on the basis of CD45RO− CCR7⁺ CD45RA⁺CD62L⁺ CD27⁺ CD11adim CD127 (7 markers) or CD45RO− CCR7⁺ CD62L⁺ (3markers). T_(SCM) were subsequently identified as CD95⁺.

FIGS. 12C-12D are graphs showing the percentage of CD8⁺ or CD4+ cellsidentified as T_(SCM) cells by multiple users on multiple days. Datawere analyzed by the same user to minimize subjectivity in the gatingprocedure **: P<0.01; ***: P<0.001.

FIGS. 13A-13F are graphs showing the expression of CCR7 (13A and 13D),CD58 (13B and 13E) or CD122 (13C and 13F) vs. CD95 in human CD4+(13A-13C) and CD8+ (13D-13F) T cells is shown as measured by flowcytometry. The gate identifies T_(SCM) cells as depicted in FIGS.11E-11V. Numbers indicate the percentage of T_(SCM) cells identified bythe gates.

FIGS. 14A-14L are graphs showing the differential response of TN (14A,14E, and 14I), T_(SCM) (14B, 14F, and 14J), TCM (14C, 14G, and 14K) andTEM (14D, 14H, 14L) (sorted as described in FIGS. 11A-11Z and stainedwith CFSE), to the stimuli anti αCD3/CD2/CD28 antibody-coated beads for6 days (14A-14D), 25 ng/mL IL-7 for 14 days (14E-14H) or 25 ng/mL IL-15for 10 days (14I-14L).

DETAILED DESCRIPTION OF THE INVENTION

An isolated population of memory T cells with enhanced stem cell-likequalities compared with the qualities of central memory T (T_(CM)) cellshas been discovered. These memory T cells, which are referred to hereinas “memory stem T cells” (T_(SCM) cells), advantageously provide anenhanced capacity for self-renewal and multipotency, and are alsocapable of repopulating differentiated effector lymphocytes in responseto antigenic stimuli. It has been discovered that T_(SCM) cells can beeffectively generated in vitro using inhibitors of glycogen synthasekinase-3β (GSK-3β). Without being bound by a particular theory ormechanism, it is believed that GSK-3β inhibitors trigger Wnt signaling,which delays or prevents T cell differentiation.

In this regard, the invention provides a method of producing an isolatedT memory stem cell population, the method comprising (a) isolating naïveT cells from a mammal, wherein the mammal is not a mouse; and (b)activating the naïve T cells and expanding the numbers of naïve T cellsin the presence of one or more non-specific T cell stimuli, one or morecytokines, and a GSK-3beta inhibitor.

The method may comprise isolating naïve T cells from a mammal by anysuitable method known in the art. For example, the naïve T cells can beobtained from the mammal by a blood draw or a leukapheresis.

Unless stated otherwise, as used herein, the term “mammal” refers to anymammal including, but not limited to, mammals of the order Logomorpha,such as rabbits; the order Carnivora, including Felines (cats) andCanines (dogs); the order Artiodactyla, including Bovines (cows) andSwines (pigs); or of the order Perssodactyla, including Equines(horses). It is preferred that the mammals are non-human primates, e.g.,of the order Primates, Ceboids, or Simoids (monkeys) or of the orderAnthropoids (humans and apes). In some embodiments, the mammal may be amammal of the order Rodentia, such as mice and hamsters. In otherembodiments, the mammal is not a mouse. Preferably, the mammal is anon-human primate or a human. An especially preferred mammal is thehuman.

The method comprises activating the naïve T cells and expanding thenumbers of naïve T cells by any suitable method known in the art. In anembodiment of the invention, the T cells are activated and the numbersof T cells are expanded in the presence of one or more non-specific Tcell stimuli and/or one or more cytokines. In an embodiment of theinvention, the T cells are activated and the numbers of T cells areexpanded by physically contacting the T cells with one or morenon-specific T cell stimuli and/or one or more cytokines. Any one ormore non-specific T cell stimuli may be used in the inventive methods.Exemplary non-specific T cell stimuli include anti-4-1BB antibodies,anti-CD3 antibodies and anti-CD28 antibodies. In preferred embodiment,the non-specific T cell stimulus may be anti-CD3 antibodies andanti-CD28 antibodies conjugated to beads. Any one or more cytokines maybe used in the inventive methods. Exemplary cytokines includeinterleukin (IL)-2, IL-7, IL-21, and IL-15.

The GSK-3beta inhibitor may be any suitable compound or composition thatinhibits GSK-3beta. Exemplary GSK-3beta inhibitors include lithiumchloride (LiCl), TWS119(3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol), BIO(6-bromoindirubin-3′-oxime), and CHIR99021(6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile).

Another embodiment of the method provides a method of producing anisolated T memory stem cell population, the method comprising (a)isolating lymphocytes from a mammal; and (b) sorting the lymphocytesusing flow cytometry into a population comprising a phenotype comprising(i) CD95+, CD45RO−, and CCR7+; and (ii) CD62L+ or one or more of CD27+,CD28+, CD45RA+, and CD127+ to produce an isolated T memory stem cellpopulation.

The method may comprise isolating lymphocytes from a mammal as describedherein with respect to other aspects of the invention. The lymphocytesmay be any lymphocytes. Preferably, the lymphocytes are nave T cells.Preferably, the mammal is a human.

The method may comprise sorting the cells in any suitable manner.Preferably, the sorting is carried out using flow cytometry. The flowcytometry may be carried out using any suitable method known in the art.The flow cytometry may employ any suitable antibodies and stains.Preferably, the flow cytometry is polychromatic flow cytometry.

The method comprises sorting the cells into a population comprising a Tmemory stem cell phenotype. The phenotype may comprise (e.g., thesimultaneous expression of) any one or more of CD95+, CD45RO−, CCR7+,CD62L+, CD27+, CD28+, CD45RA+, and CD127+. Preferably, the methodcomprises sorting the lymphocytes into a population comprising aphenotype comprising (e.g., the simultaneous expression of) (i) CD95+,CD45RO−, and CCR7+; and (ii) CD62L+ or one or more of CD27+, CD28+,CD45RA+, and CD127+. Preferably, the method comprises sorting thelymphocytes into a population comprising a phenotype comprising (e.g.,the simultaneous expression of) all of CD95+, CD45RO−, CCR7+, CD62L+,CD27+, CD28+, CD45RA+, and CD127+. Preferably, the method furthercomprises sorting the lymphocytes into a population comprising aphenotype further comprising any one or more of CD58+, CD122+, CD3+,CD4+, CD8+, CD11a^(dim) and CD11a+. Preferably, the method produces anisolated human T memory stem cell population.

Another embodiment of the invention provides a method of producing anisolated T memory stem cell population, the method comprising (a)isolating lymphocytes from a mammal; (b) sorting the lymphocytes into apopulation comprising a phenotype comprising (i) CD95+ and/or CXCR3+;and (ii) CD45RA+, CCR7+, and CD28+ using flow cytometry to produce anisolated T memory stem cell population.

The method may comprise isolating lymphocytes from the mammal asdescribed herein with respect to other aspects of the invention.Preferably, the mammal is any non-human primate. An especially preferredmammal is a rhesus macaque.

The method comprises sorting the cells into a population comprising a Tmemory stem cell phenotype. The phenotype may comprise (e.g., thesimultaneous expression of) any one or more of CD95+, CXCR3+, CD45RA+,CCR7+, and CD28+. Preferably, the method comprises sorting thelymphocytes into a population comprising a phenotype comprising (i)CD95+ and/or CXCR3+; and (ii) CD45RA+, CCR7+, and CD28+. The sorting maybe carried out as described herein with respect to other aspects of theinvention.

In an embodiment of the invention, the method further comprisesexpanding the numbers of T_(SCM) in vitro. The numbers of T_(SCM) may beincreased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), morepreferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-,or 90-fold), or most preferably at least about 100-fold. The numbers ofT_(SCM) may be expanded using any suitable method known in the art.Exemplary methods of expanding the numbers of cells are described inU.S. Pat. No. 8,034,334 and U.S. patent application Ser. No. 13/424,646,each of which is incorporated herein by reference.

In an embodiment of the invention, the method further comprisestransducing the isolated T_(SCM) with a nucleotide sequence encoding achimeric antigen receptor (CAR) or a T cell receptor (TCR) (e.g., anexogenous TCR). The CAR or TCR may have antigenic specificity for acancer antigen or a viral antigen. Exemplary CARs include thosedescribed in International Patent Application Publication No. WO2011/041093 and International Application No. PCT/US12/29861, each ofwhich is incorporated herein by reference. Exemplary TCRs include thosedescribed in U.S. Pat. Nos. 7,820,174; 8,088,379; 8,216,565; U.S. PatentApplication Publication No. 2009/0304657; and International PatentApplication Publication Nos. WO 2012/040012 and WO 2012/054825, each ofwhich is incorporated herein by reference. The cells may be transducedusing any suitable method known in the art, for example, as described inSambrook et al., Molecular Cloning: A Laboratory Manual, 3^(rd) ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons, NY, 1994.

The term “cancer antigen” as used herein refers to any molecule (e.g.,protein, peptide, lipid, carbohydrate, etc.) solely or predominantlyexpressed or over-expressed by a tumor cell or cancer cell, such thatthe antigen is associated with the tumor or cancer. The cancer antigencan additionally be expressed by normal, non-tumor, or non-cancerouscells. However, in such cases, the expression of the cancer antigen bynormal, non-tumor, or non-cancerous cells is not as robust as theexpression by tumor or cancer cells. In this regard, the tumor or cancercells can over-express the antigen or express the antigen at asignificantly higher level, as compared to the expression of the antigenby normal, non-tumor, or non-cancerous cells. Also, the cancer antigencan additionally be expressed by cells of a different state ofdevelopment or maturation. For instance, the cancer antigen can beadditionally expressed by cells of the embryonic or fetal stage, whichcells are not normally found in an adult host. Alternatively, the cancerantigen can be additionally expressed by stem cells or precursor cells,which cells are not normally found in an adult host.

The cancer antigen can be an antigen expressed by any cell of any canceror tumor, including the cancers and tumors described herein. The cancerantigen may be a cancer antigen of only one type of cancer or tumor,such that the cancer antigen is associated with or characteristic ofonly one type of cancer or tumor. Alternatively, the cancer antigen maybe a cancer antigen (e.g., may be characteristic) of more than one typeof cancer or tumor. For example, the cancer antigen may be expressed byboth breast and prostate cancer cells and not expressed at all bynormal, non-tumor, or non-cancer cells. Exemplary cancer antigens mayinclude any one or more of gp100, MART-1, MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, NY-ESO-1, vascular endothelial growth factorreceptor-2 (VEGFR-2), HER-2, mesothelin, and epidermal growth factorreceptor variant III (EGFR III).

The term “viral antigen” as used herein refers to any molecule (e.g.,protein, peptide, lipid, carbohydrate, etc.) solely or predominantlyexpressed by a virus, such that the antigen is associated with thevirus. The viral antigen can be an antigen expressed by any virus,including the viruses described herein. The viral antigen may be a viralantigen of only one type of virus, such that the viral antigen isassociated with or characteristic of only one type of virus.Alternatively, the viral antigen may be a viral antigen (e.g., may becharacteristic) of more than one type of virus. For example, the viralantigen may be expressed by a virus selected from the group consistingof herpes viruses, pox viruses, hepadnaviruses, papilloma viruses,adenoviruses, coronoviruses, orthomyxoviruses, paramyxoviruses,flaviviruses, and caliciviruses.

The inventive methods advantageously isolate T_(SCM) cells. In anembodiment, the T_(SCM) is a human T_(SCM). In an embodiment, theT_(SCM) simultaneously express multiple naïve markers including any oneor more of CD45RA, CCR7, CD62L, CD27, CD28, CD127 (IL-7Rα) andCD11a^(dim), while lacking the expression CD45RO. Unlike naïve (T_(N))cells, T_(SCM) cells also express the memory antigen CD95. In thisregard, an embodiment of the invention provides an isolated or purifiedT_(SCM) comprising a phenotype comprising (e.g., the simultaneousexpression of) any one or more of CD95+, CD45RO−, CCR7+, CD62L+, CD27+,CD28+, CD45RA+, and CD127+. Preferably, the T_(SCM) comprises aphenotype comprising (e.g., the simultaneous expression of) (i) CD95+,CD45RO−, and CCR7+; and (ii) CD62L+ or one or more of CD27+, CD28+,CD45RA+, and CD127+. Preferably, the T_(SCM) comprises a phenotypecomprising (e.g., the simultaneous expression of) all of CD95+, CD45RO−,CCR7+, CD62L+, CD27+, CD28+, CD45RA+, and CD127+. Preferably, theT_(SCM) comprises a phenotype further comprising any one or more ofCD58+, CD122+, CD3+, CD4+, CD8+, CD11a^(dim) and CD11a+.

In an embodiment, the invention also provides an isolated non-humanprimate (NHP) T_(SCM). The T_(SCM) may comprise a phenotype comprising(e.g., the simultaneous expression of) any one or more of CD95+, CXCR3+,CD45RA+, CCR7+, and CD28+. Preferably, the T_(SCM) comprises a phenotypecomprising (i) CD95+ and/or CXCR3+; and (ii) CD45RA+, CCR7+, and CD28+.

In an embodiment of the invention, the T_(SCM) comprises a CAR and/or aTCR (e.g., an exogenous TCR). The CAR and TCR may be as described hereinwith respect to other aspects of the invention.

The invention further provides an isolated or purified population ofcells comprising two or more of any of the isolated T_(SCM) cellsdescribed herein.

The term “isolated” as used herein means having been removed from itsnatural environment. The term “purified” as used herein means havingbeen increased in purity, wherein “purity” is a relative term, and notto be necessarily construed as absolute purity. For example, the puritycan be at least about 50%, can be greater than 60%, 70% or 80%, 90% orcan be 100%.

The inventive T_(SCM) cells can be included in a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the T_(SCM) cells describedherein and a pharmaceutically acceptable carrier.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used for the administration of cells. Suchpharmaceutically acceptable carriers are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one which has no detrimentalside effects or toxicity under the conditions of use.

The T_(SCM) cells may be administered in any suitable manner.Preferably, the T_(SCM) cells are administered by injection, e.g.,intravenously. A suitable pharmaceutically acceptable carrier for thecells for injection may include any isotonic carrier such as, forexample, normal saline (about 0.90% w/v of NaCl in water, about 300mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOLR electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter,Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In anembodiment, the pharmaceutically acceptable carrier is supplemented withhuman serum albumen.

For purposes of the invention, the dose, e.g., number of the inventiveT_(SCM), administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the number of the inventive T_(SCM)should be sufficient to bind to a cancer antigen, or detect, treat orprevent cancer in a period of from about 2 hours or longer, e.g., 12 to24 or more hours, from the time of administration. In certainembodiments, the time period could be even longer. The number of theinventive T_(SCM) will be determined by, e.g., the efficacy of theparticular inventive T_(SCM) and the condition of the animal (e.g.,human), as well as the body weight of the animal (e.g., human) to betreated.

Many assays for determining an administered number of the inventiveT_(SCM) are known in the art. For purposes of the invention, an assay,which comprises comparing the extent to which target cells are lysed orone or more cytokines such as, e.g., IFN-γ and IL-2 is secreted uponadministration of a given number of such T_(SCM) cells to a mammal amonga set of mammals of which is each given a different number of theT_(SCM) cells, could be used to determine a starting number to beadministered to a mammal. The extent to which target cells are lysed orcytokines such as, e.g., IFN-γ and IL-2 are secreted upon administrationof a certain number can be assayed by methods known in the art.Secretion of cytokines such as, e.g., IL-2, may also provide anindication of the quality (e.g., phenotype and/or effectiveness) of aT_(SCM) cell preparation.

The number of the inventive T_(SCM) also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive T_(SCM).Typically, the attending physician will decide the number of theinventive T_(SCM) with which to treat each individual patient, takinginto consideration a variety of factors, such as age, body weight,general health, diet, sex, route of administration, and the severity ofthe condition being treated. By way of example and not intending tolimit the invention, the number of the inventive T_(SCM) can be about10×10⁶ to about 10×10¹¹ cells per infusion, about 10×10⁹ cells to about10×10¹¹ cells per infusion, or 10×10⁷ to about 10×10⁹ cells perinfusion. The inventive T_(SCM) may, advantageously, make it possible toeffectively treat or prevent cancer by administering about 100 to about10,000-fold lower numbers of cells as compared to adoptive immunotherapyprotocols that do not administer T_(SCM).

It is contemplated that the inventive T_(SCM) cells can be used inmethods of treating or preventing cancer. In this regard, the inventionprovides a method of treating or preventing cancer in a mammal,comprising administering to the mammal any of the pharmaceuticalcompositions, T_(SCM) cells, or populations of T_(SCM) cells describedherein in an amount effective to treat or prevent cancer in the mammal.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

For purposes of the inventive methods, wherein T_(SCM) cells orpopulations of T_(SCM) cells are administered, the cells can be cellsthat are allogeneic or autologous to the host. Preferably, the cells areautologous to the host.

With respect to the inventive methods, the cancer can be any cancer,including any of sarcomas (e.g., synovial sarcoma, osteogenic sarcoma,leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g.,Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma,glioma, head-neck cancer, acute lymphocytic cancer, acute myeloidleukemia, bone cancer, brain cancer, breast cancer, cancer of the anus,anal canal, or anorectum, cancer of the eye, cancer of the intrahepaticbile duct, cancer of the joints, cancer of the neck, gallbladder, orpleura, cancer of the nose, nasal cavity, or middle ear, cancer of theoral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronicmyeloid cancer, colon cancer (e.g., colon carcinoma), esophageal cancer,cervical cancer, gastrointestinal carcinoid tumor, hypopharynx cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, ureter cancer, and urinary bladder cancer.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the identification of human T memory stemcells.

Candidate human T_(SCM) cells were generated by activating CD45RO⁻CD62L⁺naïve CD8⁺ T cells in the presence of the GSK-30 inhibitor TWS119. After2 weeks, the majority of T cells cultured with TWS119 retained aCD45RO⁻CD62L⁺ naïve-like phenotype, whereas in the absence of GSK-3βinhibition, T cells uniformly upregulated the memory marker CD45RO. Todetermine whether the CD45RO⁻CD62L⁺ T cells generated in the presence ofTWS119 were truly naïve cells or had acquired memory traits, phenotypicanalysis was performed using established markers of T cell activationand differentiation (Appay et al., Cytometry A 73: 975-983 (2008)). Thevast majority of molecules (CD45RA, CCR7, CD27, IL-2Rα, IL-7Rα, CD69,41BB, CCR5 and CD57) showed a similar expression pattern between T_(N)cells and TWS119-generated naïve-like T cells. However, the naïve-like Tcells expressed levels of CD95 and IL-2Rβ similar to those observed inmemory T cells. Thus, it was hypothesized that the expression of CD95and IL-2Rβ in otherwise phenotypically naïve T cells could identifyhuman T_(SCM) cells.

To determine if candidate T_(SCM) cells occur naturally, polychromaticflow cytometry (PFC) was used (De Rosa et al., Nat. Med., 7: 245-248(2001)). Seven markers were used to accurately define T_(N) cells.Notably, a CD95⁺IL-2Rβ⁺ subset was found inCD45RO⁻CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺IL-7Rα⁺ naïve-like CD8⁺ and CD4⁺ Tcells. In 29 healthy donors, these cells, referred to hereafter asT_(SCM) cells, represented about 2-3% of all circulating CD8⁺ and CD4⁺ Tlymphocytes (FIGS. 1A and 1B). Further phenotypic analysis of T celldifferentiation markers revealed that T_(SCM) cells also expressedhigher amounts of BCL-2, LFA-1, CXCR3, CXCR4, and lower levels of CD38and CD31 compared with T_(N) cells. T_(SCM) cells were phenotypicallydifferent from the CD161⁺, IL-18Rα⁺ cells described in Turtle et al.,Immunity, 31: 834-844 (2009) and were not mucosal-associated invariant Tcells (MAITs) based on the relative expression of RORC and IL17A(Dusseaux et al., Blood, 117: 1250-1259 (2011)) (FIGS. 1C, 1D).Similarly to memory T cells, T_(SCM) cells were detected at lowfrequencies (<1%) in umbilical cord blood. The phenotype of T_(SCM)cells suggests a tropism for lymphatic tissues.

Example 2

This example demonstrates that T_(SCM) cells possess attributes ofmemory T cells.

Because of the concomitant expression of numerous markers of naïve Tcells as well as molecules of memory differentiation, it remainedunclear whether T_(SCM) cells were functionally naïve or memory T cells.Naïve T cells have a high content of T cell receptor (TCR) rearrangementexcision circles (TRECs), which are diluted during clonal proliferation(Douek et al., Nature, 396: 690-695 (1998)). Like T_(CM) and T_(EM)cells, it was found that T_(SCM) cells had low content of TRECs,indicating that they had undergone several rounds of division (FIG. 2A).

Memory T cells can also be distinguished from T_(N) cells by theirability to rapidly acquire effector functions upon antigen rechallenge(Kambayashi et al., J. Immunol., 170: 2399-2408 (2003)). It was foundthat within 4 hours after exposure to Staphylococcus enterotoxin B(SEB), a significant fraction of CD95⁺ naïve-like CD8⁺ T cells producedIFN-γ, IL-2 and tumor necrosis factor (TNF)-α, whereas T_(N) cellsremained relatively quiescent (FIGS. 2B, 2C, and 2D). Thus, T_(SCM)cells rapidly acquired effector functions after superantigen stimulationsimilarly to memory T cells. Notably, the fraction of responding cells,as well as T cell polyfunctionality, progressively increased from T_(N)cells→T_(SCM) cells→T_(CM) cells→T_(EM) cells (FIGS. 2B, 2C, and 2D),consistent with the hypothesis that T_(SCM) cells are the leastdifferentiated memory subset. Similar findings were observed for CD4⁺ Tcells. The rapid responsiveness of T_(SCM) cells was also observed afterpolyclonal stimulation with α-CD3/CD2/CD28 beads (FIGS. 3A, 3B, and 3C).Consistent with the intracellular cytokine staining result, it was foundthat sorted T_(SCM) cells, but not T_(N) cells, secreted IFN-γ, IL-2 andTNF-α in response to α-CD3/CD2/CD28 stimulation (FIGS. 4A, 4B, and 4C).Thus, T_(SCM) cells possess the memory capability of rapid acquisitionof effector functions after TCR stimulation.

Unlike T_(N) cells, memory T cells undergo robust proliferation in thepresence of the homeostatic cytokines IL-15 and IL-7 (Surh et al., J.Exp. Med., 192: F9-F14 (2000); Prlic et al., J. Exp. Med., 195: F49-F52(2002); Lugli et al., Blood, 116: 3238-3248 (2010)). It was found that,similarly to CD8⁺ memory T cells, T_(SCM) cells divided extensively inresponse to IL-15. Although the majority of T_(EM) cells proliferated(FIG. 5A), they underwent fewer divisions, revealing a lowerproliferative potential compared with other memory subsets (FIG. 5B). Bycontrast, T_(SCM) cells underwent numerous cell divisions (FIG. 5B),although the majority of these cells remained undivided (FIG. 5A). Thisbehavior is reminiscent of stem cells, which are quiescent but can giverise to progeny capable of extensive proliferation and differentiation.Similar findings were observed in the CD4⁺ T cell compartment inresponse to IL-7. Thus, T_(SCM) cells have the replicative history andability to respond rapidly to antigenic and homeostatic stimuli, whichare characteristics of memory T cells.

The frequency of naïve CD8⁺ T cell precursors for a given epitope hasbeen estimated to be between 6×10⁻⁷ and 5×10⁻⁶, a range below the limitof peptide-major histocompatibility complex class I (pMHCI) tetramerdetection (Alanio et al., Blood, 115: 3718-3725 (2010)). It was reasonedthat if tetramer-binding, naïve-like T cells could be measured, theywould be enriched in the CD95⁺ T_(SCM) cell compartment. In donors withdetectable naïve-like CD8⁺ T cells specific to influenza orcytomegalovirus (CMV) epitopes, the vast majority of tetramer-bindingcells highly expressed CD95 (FIG. 5C). By contrast, virtually all MART-1(melanoma antigen recognized by T cells)-specific naïve-like T cells inhealthy donors did not express CD95, indicating that these cells weretruly naïve (FIG. 5C). Notably, it was found that a significant fractionof MART-1-specific CD8⁺ T cells had a CD95⁺ phenotype in 7 out of 11subjects with metastatic melanoma (FIG. 5C). Thus, tetramer-binding Tcells found in the naïve-like T cell compartment could be derived fromeither increased thymic output (CD95⁻), as reported for MART-1 inhealthy donors (Zippelius et al., J. Exp. Med., 195: 485-494 (2002)), orfrom antigenic encounter, expansion and differentiation (CD95⁺). Theseexperiments also revealed that T_(SCM) cells represented a substantialfraction of the corresponding total antigen-specific CD8⁺ T cell memoryresponses, averaging 0.6% for CMV pp 65495-503, 4.2% for influenzaM158-66 and 7.6% for MART-126-35, and that their frequency tended tocorrelate with that of memory T cells.

To determine whether T_(SCM) cell clonotypes represent a long-livedpopulation or merely recently activated cells transitioning from a naïveto a memory state, TCR-β sequences of CMV-specific T cell subsets fromthe same donor spanning a time period of 22 months were analyzed.Similarly to memory T cells, dominant persisting clonotypes in T_(SCM)cells were found, thereby indicating that they represent a stable memoryT cell population. These findings show that T_(SCM) cells are long-livedmemory T cells with multiple viral and self-tumor specificities.

Example 3

This example demonstrates that T_(SCM) cells represent the leastdifferentiated T cell memory subset.

The transcriptome of T_(SCM) cells was compared with naïve and memory Tcell subsets and findings were validated with PFC. 900 differentiallyexpressed genes were found among the four CD8⁺ T cell subsets (P<0.01,false discovery rate<5%). Unsupervised hierarchical clustering revealedthat T_(SCM) cells had a distinct gene expression profile more closelyrelated to that of memory T cells than of T_(N) cells, furthercorroborating the idea that T_(SCM) cells are a unique T cell memorysubset. Consistent with previous findings (Willinger et al., J.Immunol., 175: 5895-5903 (2005)), the expression of the majority ofgenes (565 of 900) progressively increased (effector-associated genes)or decreased (naïve-associated genes) in the exact order: T_(N)cells→T_(SCM) cells→T_(CM) cells→T_(EM) cells. Transcripts encodingregulators of effector differentiation and senescence, such aseomesoderminutes (Pearce et al., Science, 302: 1041-1043 (2003)), T-box21 (Joshi et al., Immunity, 27: 281-295 (2007)) and PR domain containing1 with ZNF domain (Rutishauser et al., Immunity, 31: 296-308 (2009)), aswell as cytotoxic molecules (for example, granzyme A and perforin) andmarkers of T cell senescence (for example, killer cell lectin-likereceptor subfamily G, member 1, KLRG1) (Joshi et al., Immunity, 27:281-295 (2007)), were increasingly expressed from T_(N) cells to T_(EM)cells (FIGS. 6A-6L). Conversely, transcripts encoding transcriptionfactors that inhibit T cell activation and differentiation, includinglymphoid enhancer-binding factor 1 (Gattinoni et al., Nat. Med., 15:808-813 (2009)), forkhead box P1 (Feng et al., Nat. Immunol., 12:544-550 (2011)), and LAG1 homolog, ceramide synthase 6, which promotescellular quiescence by regulating intracellular ceramide levels(Ogretmen et al., Nat. Rev. Cancer, 4: 604-616 (2004)), progressivelydecreased from T_(N) cells to T_(EM) cells (FIGS. 6A-6L). These data areconsistent with a linear model of T cell differentiation, in whichT_(SCM) cells are the least differentiated memory T cell subset.

Multidimensional scaling (MDS) analysis (Khan et al., Cancer Res., 58:5009-5013 (1998)) confirmed that T_(SCM) cells comprised the memory Tcell subset most similar to T_(N) cells. Indeed, it was found that only75 genes were differentially expressed between T_(N) and T_(SCM) cells(P<0.01 and >twofold change in expression) compared with 157 and 226 forT_(CM) and T_(EM) cells, respectively. T_(SCM) and T_(CM) cells were themost closely related T cell subsets, with 20 differentially expressedgenes. Among these genes, T_(SCM) cells, like T_(N) cells, expressed lowamounts of HNRPLL (encoding heterogeneous nuclear ribonucleoproteinL-like), a regulator of the alternative splicing of the CD45 pre-mRNArequired for efficient CD45RO expression (Oberdoerffer et al., Science,321: 686-691 (2008)), thus confirming the purity of the sorting. Whenthis subset of 20 genes was considered, it was found that T_(SCM) cellshad a pattern of expression similar to that of T_(N) cells, whereasT_(CM) cells clustered with T_(EM) cells, further underscoring thenotion that T_(SCM) cells are less differentiated than T_(CM) cells.

Example 4

This example demonstrates the enhanced self-renewal and multipotency ofT_(SCM) cells.

The abilities to self-renew and to differentiate into specialized celltypes are qualities of stem cells. To determine whether T_(SCM) havethese stem cell-like properties, their capacity to self-renew withhomeostatic signals as well as their multipotency after TCR activationwere evaluated. After exposure to IL-15, the vast majority of T_(SCM)cells maintained CD45RA⁺, and retained significantly (P<0.05) higheramounts of CD62L and CCR7 than T_(CM) cells (FIGS. 7A and 7B). At theend of stimulation, about 60% of cells derived from T_(SCM) maintainedtheir phenotypic identity (CCR7⁺CD62L⁺CD45RA⁺CD45RO⁻), but only 30% ofTCM cells retained their input phenotype (CCR7⁺CD62L⁺CD45RA⁻CD45RO⁺)(FIG. 7C). T_(SCM) cells also showed greater self-renewal capacitycompared with T_(CM) cells after a secondary exposure to IL-15 (FIG.7D).

After α-CD3/CD2/CD28 stimulation, however, T_(SCM) cells graduallyupregulated CD45RO over several cell divisions while acutelydownregulating CD62L and CCR7 (FIGS. 8A and 8B). These dynamic changesin phenotype resulted in a diverse progeny, comprising about 50% ofT_(CM) cells and 4% of T_(EM) cells. Most notably, 15% ofT_(SCM)-derived cells maintained a CCR7⁺CD62L⁺CD45RA⁺CD45RO⁻ phenotypeeven after this potent stimulus, thus indicating that T_(SCM) cells havethe multipotent capacity to derive all memory T cell subsets. Bycontrast, it was found that T_(CM) cells retained a central memoryphenotype or differentiated into T_(EM) cells, but they did not generateT_(SCM) cells. Consistent with their advanced differentiation state,T_(EM) cells did not reacquire CD62L or CCR7 and did not dedifferentiateinto T_(CM) or T_(SCM) cells after either IL-15 or α-CD3/CD2/CD28stimulation (FIGS. 7A-7C and 8A-8B). Taken together, these findingssuggest that T_(SCM) cells have the stem cell-like properties ofself-renewal and multipotency in vitro (FIG. 8C).

Example 5

This example demonstrates the increased proliferative capacity, survivaland antitumor activity of T_(SCM) cells.

To evaluate the replicative responses of T_(SCM) cells, ³H-thymidineincorporation after TCR stimulation was measured. T_(CM) and T_(N) cellsshowed increased proliferative responses compared with T_(EM) cells, butthey were outpaced by T_(SCM) cells (FIG. 9A). The long-term replicativeand survival capacities of T_(SCM) cells was ascertained. CD8⁺ T cellsubsets were adoptively transferred into highly immunodeficientNOD.Cg-Prkdc^(scid)Il2rg^(tm1wjl)/SzJ (NSG) mice and T cell engraftmentwas evaluated 1 month after transfer. CD8-depleted peripheral bloodmononuclear cells (PBMCs) were co-transferred to provide a source ofhuman cytokines and co-stimulatory molecules (Carpenito et al., Proc.Natl. Acad. Sci. USA, 106: 3360-3365 (2009)). It was found that T_(SCM)cells engrafted with 10- to 100-fold more progeny than T_(CM) or T_(N)cells in both lymphoid and nonlymphoid tissues (FIGS. 9B-9G). Notably,T_(EM) cells, which resemble cell populations used in current clinicaltrials for adoptive immunotherapy (Gattinoni et al., Nat. Rev. Immunol.,6: 383-393 (2006); June et al., J. Clin. Invest., 117: 1466-1476(2007)), had a poor proliferative and survival capability resulting innegligible engraftment one month after transfer (FIGS. 9B-9G). Althoughthe CD8⁺ T cell subsets differentiated into effector cells, perhaps as aresult of encounter with homeostatic cytokines and with xenogeneic majorhistocompatibility antigens, the adoptive transfer in NSG mice suggeststhat T_(SCM) cells have enhanced replicative and survival capabilitiescompared with naïve and memory subsets.

T cell proliferative and survival capacities correlate with theantitumor efficacy of adoptively transferred T cells (Gattinoni et al.,Nat. Rev. Immunol., 6: 383-393 (2006); June et al., J. Clin. Invest.,117: 1466-1476 (2007); Gattinoni et al., J. Clin. Invest., 115:1616-1626 (2005); Klebanoff et al., Proc. Natl. Acad. Sci. USA, 102:9571-9576 (2005); Hinrichs et al., Proc. Natl. Acad. Sci. USA, 106:17469-17474 (2009)). TCR or chimeric antigen receptor (CAR) geneengineering may be used in the clinic to redirect the specificity ofcirculating T cells toward the desired target (Morgan et al., Science,314: 126-129 (2006); Pule et al., Nat. Med., 14: 1264-1270 (2008)). Thisapproach was exploited, coupled to the pharmacological activation of Wntsignaling, to generate high numbers of mesothelin-specific exvivo-generated memory T cell subsets to test in a xenograft tumor modelthat we recently established (Carpenito et al., Proc. Natl. Acad. Sci.USA, 106: 3360-3365 (2009)). Mesothelin-specific T_(SCM), T_(CM) orT_(EM) cells were co-transferred with mesothelin-specific CD4⁺ T cellsinto NSG mice bearing luciferase-expressing M108 mesotheliomaestablished for 3 months in the peritoneum. To generate a treatmentwindow, 3×10⁶ CD8⁺ T cells and 10⁶ CD4⁺ T cells were administered, about10% of the previously described curative dose in this humanized tumormodel (Carpenito et al., Proc. Natl. Acad. Sci. USA, 106: 3360-3365(2009)). T_(EM) cells mediated poor antitumor responses, as indicated bythe high intensity of the tumor-derived bioluminescent signal in theabdomen and the ascites-dependent weight gain (FIG. 10A). Furthermore,the transfer of T_(EM) cells did not significantly extend the survivalof the mice compared with CD4⁺ T cells alone (FIG. 10B). T_(CM) cellswere more effective than T_(EM) cells but all mice died from tumorprogression within 40 days after treatment (FIGS. 10A-10B). In contrast,T_(SCM) cells triggered tumor regression and cure in mice that otherwisedied within 2-3 weeks in the absence of CD8⁺ T cell transfer (FIGS.10A-10B). The late mortality of mice receiving T_(SCM) cells wasascribed to the development of xenogeneic graft-versus-host disease, asmanifested by loss of body weight (FIG. 10A). These data suggest thatadoptively transferred T_(SCM) cells have enhanced antitumor activityand are more therapeutically effective than T_(CM) and T_(EM) cells inmice.

Example 6

This example demonstrates a method of isolating a memory stem cellpopulation.

Human and non-human primate (NHP) T_(SCM) cells are relatively rare,comprising about 2-4% of total CD4⁺ or CD8⁺ T cells in the blood. Bypolychromatic flow cytometry, human T_(SCM) were characterized assimultaneously expressing multiple naïve markers including CD45RA, CCR7,CD62L, CD27, CD28, CD127 (IL-7Rα) and CD11a^(dim) and lacking CD45RO;unlike naïve (T_(N)) cells, they also express the memory antigen CD95.However, the simultaneous analysis of all of these nine markers is notnecessary for the identification of human T_(SCM) cells (FIGS. 12A and12B). 7- or 8-color panels (Table 1) accurately identify and allow forsorting of human and NHP T_(SCM) using commonly-available flowcytometers. All antibody/fluorochrome combinations described arecommercially available.

TABLE 1 Panel # 1 2 3 Target cell Human T_(SCM) (bulk or Human T_(SCM)NHP T_(SCM) antigen-specific) (bulk or antigen- specific) Source ofFresh Cryopreserved Fresh or cells Cryopreserved AmCyan AQUA fluorescentAQUA Live/ AQUA Live/ reactive dye Dead fluorescent Dead fluorescentreactive dye reactive dye APC-Cy7 CD3 CD3 CD3 Pacific CD4 or CD8 CD4 orCD8 CD4 or CD8 Blue APC CD45RO CD45RO CD95 FITC CCR7 CCR7 CCR7 PE-Cy7CD62L CD27 CD45RA PE-Cy5 CD95 CD95 ECD — — CD28 PE MHC class I tetramerMHC class I CXCR3 CD58 tetramer CD122 CD58 CD122

For human cells (Table 1, panels #1 and #2), the panels include: i) a“dump” channel to exclude dead cells with a viability dye; ii)antibodies to CD3, CD8 and CD4 to define the lineage of interest; iii)antibodies to CD45RO, CCR7 and either CD62L or a different markerexpressed by nave cells (e.g. CD27, CD28 or CD45RA) to identifynaïve-like cells and subsets of memory cells (De Rosa et al., Nat. Med.,7: 245-248 (2001)); iv) and anti-CD95 to discriminate CD95⁻ T_(N) fromCD95⁺ T_(SCM). These panels leave the PE channel open to accommodate anadditional antibody of interest, an MHC class I tetramer for theidentification of antigen-specific CD8⁺ T cells, or anti-CD58 oranti-CD122 (FIGS. 13A-13F). CD58, the lymphocyte function-associatedantigen (LFA)-3, belongs to the immunoglobulin superfamily and mediatesthe interaction of lymphocytes to CD2, expressed on a variety of celltypes including the endothelium. CD122 is the β chain of the IL-2/IL-15receptor complex which forms a low affinity receptor together with the γchain. Both CD58 and CD122 are found at higher levels on memory cellsand T_(SCM) cells than on T_(N). This differential expression isutilized to better identify T_(SCM) cells (FIGS. 13A-13F). Anti-CD95antibodies are available through multiple vendors and as conjugated todifferent fluorochromes, thus allowing the design of complex andinterchangeable multicolor panels. Staining for CD95 provides a betterseparation of T_(SCM) cells from T_(N) by flow cytometry compared toCD58 and CD122. However, the vast majority of CD95⁺ T_(SCM) cells alsoco-expresses these markers. T_(SCM) cells identified on the basis of theincreased expression of CD58 or CD122 have also increased expression ofthe T_(SCM) core phenotypic marker CD95, thus indicating that CD58 andCD122 are valid markers for the identification of true T_(SCM) cells.

Anti-CD95 clone DX2 antibody (like other anti-CD95 antibodies) iscapable of inducing apoptosis in target cells. Although quiescentlymphocytes are generally resistant to CD95-induced apoptosis (Schmitzet al., J. Immunol. 171: 2930-2936 (2003); Lugli et al., Leuk. Res., 33,140-150 (2009)), sodium azide (NaN₃) is included in the staining bufferand the sample is kept cold during long FACS sorting procedures tominimize cellular metabolism.

When quantifying T_(SCM) cells in patient samples, peripheral bloodlymphocytes from a healthy donor are included as a control to help setgates. It is found that T cells from patients with different pathologiesor receiving different therapies exhibit substantially alteredrepresentation of the subsets making it difficult to judge delineationgates “by eye.”

A similar combination of antibodies is used to identify NHP T_(SCM)cells, i.e. CD45RA, CCR7, CD28, CD95 and CXCR3 (Table 1, Panel #3).

If flow cytometry sorting is planned, cell staining is preceded bynegative magnetic isolation of the target lineage (CD4+ or CD8+) toshorten the sorting time. Sorted cells are subsequently expanded bystimulating with a combination of IL-7, which preferentially expandsT_(N), T_(SCM) and T_(CM), IL-15, which selectively expands memory cells(Lugli et al., Blood, 116: 3238-48 (2010)), or anti-CD3/CD2/CD28antibody-coated beads. In contrast to the latter, IL-7 and IL-15mediated expansion partly maintains the initial phenotype of thepopulation (Geginat et al., J. Exp. Med., 194: 1711-1719 (2001); Geginatet al., Blood, 101: 4260-4266 (2003)) rather than inducing excessiveproliferation, acquisition of effector function and in vitro-inducedsenescence (Gattinoni et al., J. Clin. Invest., 115: 1616-1626 (2005)).

Human T_(SCM) cells are identified as expressing multiple markers ofT_(N) but also CD95, which is preferentially found on the surface ofmemory cells. It was found that three markers, i.e. CD45RO, CCR7 andCD62L, are sufficient for the identification of T_(N)-like cells(defined as CD45RO−CCR7⁺ CD62L⁺) and for the exclusion of memory T cellcontaminants of unknown function, as can occur when only two markers areused to identify naïve T cells (De Rosa et al., Nat. Med., 7: 245-248(2001)). Even though statistically significant differences were found,the proportion of CD8⁺ and CD4⁺ T_(SCM) cells changes only minimallywhen T_(N)-like cells are defined on the basis of 7 (mean±SEM CD8⁺:2.95±0.56; CD4⁺: 2.81±0.38) vs. 3 markers (CD8⁺: 3.40±0.62; CD4⁺:3.59±0.45; FIG. 12A). Without being bound by a particular theory ormechanism, it is believed that these differences may be ascribed, atleast in part, to the transition from CD45RA to CD45RO expression notbeing as complete as for other memory-defining antigens, thus some cellswith intermediate expression of CD45RA but negative for CD45RO might beincluded in the final population. If using a flow cytometer with alimited number of detectors (e.g. 8), the use of anti-CD45RO ispreferred instead of anti-CD45RA, as anti-CD45RO allows the exclusion ofCD45RO⁺/CD45RA⁺ activated cells. However, if more detectors areavailable, additional inclusion of anti-CD45RA better delineates thehuman T_(SCM) cells. When cryopreserved cells are used, CD62L stainingmay not be reliable because expression may be lost with the freeze/thawprocedure (Perfetto et al., Nat. Rev. Immunol., 4: 648-655 (2004)). Inthis case, a different marker is used for the identification ofnave-like cells, such as, e.g., CD27, CD28 or CD127 (IL-7Rα), asdepicted in Table 1 (Panel #2).

A standardized gating strategy is developed (FIGS. 11A-11V). It is notedthat T_(SCM) cells (especially in humans) have slightly lower levels ofCCR7 compared to T_(N) cells. This property allows better delineation ofT_(N) and T_(SCM) cells when CCR7 is plotted against CD95 expression andis exploited by positioning the sorting gate on a diagonal alongside theT_(SCM) population (FIGS. 11A-11D). Indeed, clear-cut separation ofpositive and negative expression of CD95 is visualized when using thisapproach. The gate identifying CD95+ cells is then copied in the samebivariate plot after gating for multiple nave markers, i.e. CD45RO, CCR7and CD62L/CD27 for human cells and CD45RA, CCR7 and CD28 for rhesusmacaques (FIGS. 11A-11V). Negligible differences are observed betweenexperiments performed by different users and in different days by usingthe mentioned strategy (FIGS. 12C-12D).

Alternatively, the inclusion of more markers in the panel improvesseparation of the T_(SCM) population; for example, higher levels of CD58and CD122 are found on T_(SCM) cells compared to T_(N) cells (FIGS.13A-13F). Anti-CD58 or anti-CD122 antibodies are available conjugated toPE and are included in both Panel #1 and Panel #2. More complex panelsare developed as described in Mahnke et al., Clin. Lab. Med., 27:469-485 (2007).

A similar combination of antibodies is used to track T_(SCM) cells inrhesus macaques, based on expression of CD45RA, CCR7, CD28 and CD95. InNHP, CD95 expression in the T_(SCM) vs. T_(N) populations is not asdistinct as in humans (FIGS. 11E-11V). Addition of anti-CXCR3 to thepanel improves identification of T_(SCM) cells in CD8⁺, but not the CD4⁺T cells lineage, as all NHP CD8⁺ but not CD4⁺ T_(SCM) express CXCR3(FIGS. 11E-11V). Indeed, CXCR3 replaces CD95 for the identification andisolation of CD8⁺ T_(SCM) cells in NHP (FIGS. 11E-11V).

Fluorescence Minus One (FMO) controls, i.e., samples stained with allfluorochromes except the one of interest (Perfetto et al., Nat. Rev.Immunol., 4: 648-655 (2004)), may not be fully informative in thisparticular example, to identify CD95+ cells, as some T_(N) express lowlevels of CD95 (FIGS. 11W-11Z). These cells are not included the T_(SCM)cell gate due to the difficulty of clearly separating CD95^(dull) andCD95⁻ T_(N) cells. Clear-cut separation of CD95 expression is easilyvisualized by plotting CD95 vs. CCR7, as described above. However, FMOcontrols are used to guide the gating procedure and to revealcompensation artifacts.

The flow cytometer is setup as described in Perfetto et al., Nat.Protoc. 1: 1522-1530 (2006). The flow cytometer setup includes settingdetector photomultiplier (PMT) voltages in advance using quality controlreagents, such as pre-stained beads, thereby ensuring the greatestsignal-to-background separation. Using such procedures, no changes inPMT settings are needed before the initiation of the experiment, thusthe time spent at the machine is limited to the acquisition of thesample. Quality control of laser alignment, laser delays and PMTtransmission is checked before every experiment by running Rainbowbeads, as described (Perfetto et al., Nat. Protoc. 1: 1522-1530 (2006)).In order to minimize the time spent at the flow cytometer on the day ofthe experiment, the experiment template is set up in advance. A rigorousquality assurance/quality control (QA/QC) program for instrumentalignment and settings facilitates reproducible evaluation of T_(SCM)cells using polychromatic flow cytometry.

The MHC class I tetramer is synthesized and conjugated in thelaboratory. All antibodies and tetramers are carefully titrated beforeuse, whether obtained commercially or synthesized in the laboratory. Thetitre giving the best separation over the background is chosen. However,in some cases, a lower concentration of the antibody is used to minimize“spreading error” to other fluorochromes (i.e., after compensation)(Roederer et al., Cytometry, 45: 194-205 (2001)). Detailed theoreticalconsiderations and practical procedures regarding antibody binding toantigen for flow cytometric analyses are carried out as described inKantor et al., Handbook of Experimental Immunology, Vol. 49: 1-13(Blackwell Science, Herzenberg et al. (1997)).

Example 7

This example demonstrates the identification, isolation and in vitroexpansion of human T_(SCM) cells.

Reagents

Human peripheral blood or NHP peripheral blood, lymph node or spleen

Ficoll-Paque PLUS (GE Healthcare, Pittsburgh, Pa.)

Dulbecco's Phosphate Buffered Saline (DPBS; Life Technologies, GrandIsland, N.Y.)

Heat-inactivated Fetal Bovine Serum (HI FBS; Life Technologies)

Penicillin-Streptomycin-Glutamine (Life Technologies)

RPMI 1640—phenol red (Life Technologies)

RPMI 1640—no phenol red (Life Technologies)

HEPES (Life Technologies)

Sodium Azide (NaN₃; Sigma-Aldrich)

CD4⁺ T cell isolation kit II (Miltenyi Biotech, Auburn, Calif.)

CD8⁺ T cell isolation kit (Miltenyi Biotech)

CD4⁺ T cell isolation kit, nonhuman primate (Miltenyi Biotech)

CD8⁺ T cell isolation kit, nonhuman primate (Miltenyi Biotech)

Fluorescently-conjugated anti-human monoclonal antibodies (all listedantibodies are whole immunoglobulin): anti-human CD3 APC-H7 (BDPharmingen, San Diego, Calif.; clone SK7; IgG₁κ), anti-human CD4Brilliant Violet 421 (Biolegend, San Diego, Calif.; clone OKT4;IgG_(2b κ)); anti-human CD8 Pacific Blue (BD Pharmingen; clone RPA-T8;IgG_(1κ)); anti-human CD4SRO APC (BD Pharmingen; clone UCHL1;IgG_(2aκ)); anti-human CCR7 FITC (BD Pharmingen; clone 150503;IgG_(2aκ)); anti-human CD62L PE-Cy7 (Biolegend; clone DREG-56; IgG₁κ);anti-human CD27 PE-Cy7 (Beckman Coulter, Indianapolis, Ind.; clone1A4CD27; IgG_(1κ)); anti-human CD95 PE-Cy5 (Biolegend; clone DX2;IgG_(1κ)); anti-human CD3 APC-Cy7 (BD Pharmingen; clone SP34-2; IgG₁ λ);anti-human CD45RA PE-Cy7 (BD Biosciences; clone L48; IgG_(1κ));anti-human CD28 ECD (Beckman Coulter; clone CD28.2; IgG₁); anti-humanCD95 APC (BD Pharmingen; clone DX2; IgG_(1 κ)); anti-human CXCR3 PE (BDPharmingen; clone 1C6/CXCR3; IgG_(1κ)); anti-human CD58 PE (BDBiosciences; clone L306.4; IgG_(2aκ)); anti-human CD122 PE (BDPharmingen; clone Mik-β3; IgG_(1κ)). Staining combinations are asdescribed in Table 1. Each lot of antibody is titrated before use

MHC Class I tetramers: NIH tetramer core facility

Mouse anti-monkey CD3 antibody (Life Technologies; clone FN18; IgG₁)

Mouse anti-human CD28 antibody (BD Biosciences; clone CD28.2;IgG_(1, κ))

LIVE/DEAD AQUA fluorescent reactive dye (Life Technologies)

Each lot of dye is titrated before use.

BD CompBeads anti-mouse Igκ (BD Biosciences)

SPHERO™ COMPtrol goat anti-mouse Ig (H⁺L) particles (Spherotech, LakeForest, Ill.)

R-NH₂ Beads (SMPLX Amine active beads; Bangs Laboratories, Fishers,Ind.)

Formaldehyde, 20% aqueous (Tousimis, Rockville, Md.)

Carboxy-Fluorescein di-acetate Succinimidyl Esther (CFSE; LifeTechnologies)

Recombinant human Interleukin-7 (Peptrotech, Rocky Hill, N.J.)

Recombinant human Interleukin-15 (Peptrotech)

Human T cell activation and expansion kit (Miltenyi Biotech)

Ethidium Bromide (EB; Life Technologies).

Acridine Orange (AO; Life Technologies)

Equipment

5 mL polystyrene round-bottom tubes (BD Falcon, Bedford, Mass.)

15 mL conical tubes (BD Falcon)

50 mL conical tubes (BD Falcon)

1.5 mL Eppendorf tubes (Eppendorf, Hamburg, Germany)

Quadro MACS Starting Kit (Miltenyi Biotec)

Miltenyi LS columns (Miltenyi Biotec)

Flow cytometer or cell sorter equipped with a violet, a blue and a redlaser, capable to collect 8 different fluorescences

Tissue culture 6-well plates (Corning, Corning, N.Y.)

Tissue culture 24-well plates (Corning)

Tissue culture 96-well plates (Corning)

Bench-top Ultrasonic Cleaner (Branson, Danbury, Conn.)

Cellometer automated cell counter (Nexcelom Bioscience, Lawrence, Mass.)

Cellometer disposable counting chambers (Nexcelom Bioscience)

Reagent Setup

Complete culture medium (R10): R10 is prepared using 10% (vol/vol) FBS,1% (vol/vol) Penicillin/Streptomycin/L-Glutamine in RPMI 1640 mediumwith phenol red.

Staining Buffer I: 4% (vol/vol) FBS in RPMI 1640 medium with no phenolred

Staining Buffer II: 4% (vol/vol) FBS, 0.02% (vol/vol) NaN3 in RPMI 1640medium with no phenol red

Sorting Buffer: 4% (vol/vol) FBS and 25 mM HEPES in RPMI 1640 mediumwith no phenol red.

MACS buffer: 5 mL of 0.5 M EDTA stock (5 mM) and 2.5 g BSA is mixed andPBS is added to adjust the volume to 500 mL. The solution isfilter-sterilized and degassed.

EB stock solution: 3 mg/mL of EB is mixed in ethanol and stored in darkbottle for 6 months.

AO stock solution: 5 mg/mL of AO is mixed in ethanol and stored in darkbottle for 6 months.

EB/AO working solution: 10 μL of EB stock is added to 10 μL of AO stockand diluted to 1 mL with PBS. (Final concentration (conc.) of EB=30μg/mL and final conc. AO=50 μg/mL).

AQUA viability dye: AQUA powder is thawed at 37° C. for 30 sec, 50 μL ofDMSO is added. The mixture is pipetted thoroughly and stored at −20° C.for up to 3 months.

Bead medium: 2% FBS, 0.02% NaN₃ in PBS.

R-NH₂ AQUA Compbeads: 1:5 dilution of the bead stock is made with beadmedium (approximately 46.2×10⁶ beads/mL), 350 μL (16×10⁶) is taken andwashed in PBS, and beads are resuspended in 300 μL of PBS. 100 μL AQUADye is added and incubates for 1.5 hours (hr.). Beads are washed 2× withbead medium and resuspended in 2 mL volume. Equal concentration (350 μL)of unstained amine bead is spiked in. Bead media is added to obtain afinal volume of 4 mL.

CFSE stock: powder is thawed and resuspended in DMSO at a finalconcentration of 5 mM.

Anti-monkey CD3 stimulation solution: Antibody is diluted to a finalconcentration of 10 μg/mL in PBS.

Cell Isolation and Staining.

Timing: 2 hr for Ficoll separation, 2 hr for magnetic separation, 1.5 hrfor fluorescent staining. Lymphocytes are isolated from peripheral bloodor from a different site of acquisition by Ficoll gradientcentrifugation according to standard techniques. If working with frozencells, cells are thawed according to standard techniques.

Cell number and viability are determined with CELLOMETER VISIONautomated cell counter: 20 μL of EB/AO working solution is added to 20μL of cell suspension and counted.

At least 0.5×10⁶ cells are used for simple phenotyping and 4×10⁶ cellsare used for the analysis of antigen-specific T_(SCM) cells. If sortingis planned, enough cells are started with to obtain the desired numberof T_(SCM) cells. The yield after sorting is 1 T_(SCM) cell per 250peripheral blood mononuclear cells (PBMC) for CD4⁺ T cells and 1 T_(SCM)cell per 500-1,000 PBMC for CD8⁺ T cells, depending on the donor.Similar numbers are obtained for NHP T_(SCM) cells. Optionally, if flowcytometry sorting is planned, CD4⁺ or CD8⁺ T cell populations areenriched by negative selection with a kit according to themanufacturer's instructions. If performing simple phenotyping or sortingsmall numbers of T_(SCM) cells, thawing and staining are performed onthe same day. If fixed, samples are run the following day. If aconsiderable number (millions) of T_(SCM) cells are needed, such as foradoptive transfer experiments, flow cytometric sorting may take manyhours. Enriched cells are left at 37° C. overnight and surface stainingis performed the following day, before sorting. If analyzing mRNAexpression by gene array, cells are recovered without interruption andkept at 4° C. to avoid changes in gene expression.

The cells are washed with PBS to remove any residual proteins. The cellsuspension is centrifuged for 5 minutes at 400 g at room temperature(RT). An AQUA dye working solution is prepared in excess (15% more thanthe volume needed for the experiment) by diluting the stock solution inwater and vortexing. PBS is added to reach the desired concentration asdetermined by titration and vortexed. The supernatant is removed frompelleted cells.

AQUA dye working solution is added to the cell pellet, resuspended bypipetting and incubated for 15 minutes at RT in the dark. 100 μL of AQUAdye is used if up to 10×10⁶ cells are stained. If more cells are used,it is considered that a 100×10⁶ cell pellet corresponds to a volume of˜100 μL. If 100 μL of staining solution are used to stain such a numberof cells, the final concentration of the dye (or of the antibody) isdiluted. Therefore, a staining solution is prepared containing 3× or 4×the concentration of the reagent to obtain a final volume of ˜200 uL.The staining volume is scaled up accordingly to the number of cells. Inany case, the optimal titer of antibodies to be used in sortingexperiments is determined by a titration experiment, where for instance1×, 2×, 4× or 8× the amount of the antibody optimal for staining 10⁶cells is used. Detailed theoretical considerations and practicalprocedures regarding antibody binding to antigen for flow cytometricanalyses are performed as described in Kantor et al., Handbook ofExperimental Immunology, 49: 1-13 (Blackwell Science, Herzenberg et al.,(1997)).

The cells are washed by adding Staining Buffer I (to dilute stainingsolution by 20-30 fold). The cells are spun for 5 minutes at 400 g atRT.

CCR7 staining solution is prepared in excess (15% more than the volumeneeded for the experiment) in FACS buffer I. CCR7 as well as otherchemokine receptors recycle through the plasma membrane. NaN₃ is notincluded in the staining buffer as it prevents the internalization ofsurface antigens and produces a loss of fluorescence intensity. Theamount of antibody needed to stain a large number of cells is determinedas described above.

Antibody aggregates are removed by spinning the solution in a microfugeat 15,000 g for 5 min. Only the supernatant is collected. Thesupernatant is removed from the pelleted cells.

CCR7 staining solution is added, the cell pellet is resuspended bypipetting and incubated for 20 minutes at 37° C. in the dark. If usingNHP cells, CXCR3 staining is performed at this stage. Incubation at 37°C. allows CCR7 and CXCR3 to recycle through the plasma membrane andimproves their detection by producing a gain of fluorescence. However,for rhesus macaques, no difference is seen by staining for CCR7 at 37°C. vs. RT.

The cells are washed with Staining Buffer II and spun for 5 minutes at400 g at RT. In the meantime, surface staining antibody mix is preparedin excess (15% more than the volume needed for the experiment) andcentrifuged at 15,000 g for 5 min. This mix is prepared using StainingBuffer II containing NaN₃ to minimize cellular metabolism.

The supernatant is removed from pelleted cells. Mix is added to the cellpellet, resuspended by pipetting and incubated for 20 minutes at RT inthe dark. The amount of antibody needed to stain a large number of cellsis determined as described above.

Compensation controls are prepared. Beads are vortexed and 30 μL isaliquoted to each tube. A tube is prepared for each fluorochrome plus atube with beads only (unstained negative control). If using differenttypes of beads or cells for compensation, the relative negative controlis included. Compbeads tend to form aggregates over time. Before use,Compbeads are sonicated for 2 min.

The fluorescently-conjugated antibody is added at the same titer that isused for the staining, vortexed and incubated for 15 minutes at RT. Thesample and compensation controls are washed with Staining buffer II andspun for 5 minutes at 400 g at RT.

If performing phenotyping, cells are resuspended in 1% PFA solution. Ifsorting, cells are resuspended in Sorting Buffer. Cells are kept on iceand in the dark. Compensation tube contents are resuspended in the samebuffer. If performing a long sort, cells are resuspended in RPMI 1640medium supplemented with HEPES. Indeed, CO₂-based buffers lose pH underhigh sort pressures, thus reducing cell survival after sort.

Acquisition and Cell Sorting.

Timing: ˜2-15 h, depending on the number of samples and cells requiredfor the experiment. Compensation controls are run. Compensation matrixis created and applied to tubes, if sorting. Run the samples using thegating strategy shown in FIGS. 11A-11Z.

Using a 70 μm nozzle, the population(s) of interest are sorted: humanT_(N) (CD45RO⁻CCR7⁺ CD62L⁺ CD95−); human T_(SCM) (CD45RO⁻CCR7⁺ CD62L⁺CD95⁺); human TCM (CD45RO⁺ CCR7⁺); human T_(EM) (CD45RO⁺ CCR7⁻); NHPT_(N) (CD45RA⁺ CCR7⁺ CD28⁺ CD95⁻); NHP T_(SCM) (CD45RA⁺ CCR7⁺ CD28⁺CD95⁺); NHP T_(CM) (CD45RA⁻CCR7⁺); NHP T_(EM) (CD45RA⁻CCR7⁺). The cellsare sorted into a 5 mL Falcon tube or 1.5 mL Eppendorf tube containingR10 complete medium, if cell culture is planned afterwards. 250,000T_(SCM) cells/h are obtained by flow cytometry sorting. Sorted cells arekept chilled to minimize cellular metabolism. However, the sample is notchilled if a short stimulation is planned, to avoid cellnon-responsiveness. The purity of sorted subsets is checked to be >95%.

T Cell Expansion In Vitro.

Timing: 7-14 days. If stimulating NHP cells, a plate is coated withanti-monkey CD3 overnight at 4° C. as described above. The antibodysolution is removed and washed three times with cold PBS before addingcell suspension. The cells are washed in R10 if proceeding directly tocell culture and stimulation. If performing CFSE staining to track cellproliferation, the cells are washed with PBS to remove any traces ofproteins. The cells are pelleted by centrifuging for 5 minutes at 400 gat RT.

Optionally, the cells are stained with CFSE. The CFSE working solutionis prepared by adding 2 μL of the stock to 1 mL of PBS (finalconcentration 10 μM). The working solution is pre-warmed at 37° C.before adding to the cell pellet. The supernatant is removed from thecell pellet. The appropriate volume of CFSE is added to achieve ˜10⁷cells/mL and vortexed. The sample is incubated for 7 minutes in a 37° C.water bath. 1-2 mL cold FBS is added to stop the reaction, vortexed, andtopped up with R10. The tubes are centrifuged for 5 minutes at 400 g.The supernatant is removed and resuspended in R10 at a density of2.5×10⁵ cells/mL.

The cells are cultured in the presence of the appropriate stimuli. Someextra wells are left with unstimulated CFSE-stained cells to be used asa compensation control at the time of analysis. Unstained PBMC providean appropriate negative control. Human T cell subsets are efficientlyexpanded with anti-CD3/CD2/CD28 beads, IL-7 or IL-15, as depicted inFIGS. 14A-14L. NHP T cell subsets are expanded by stimulating withplate-bound anti-CD3 and soluble CD28 (final concentration: 1 μg/mL).Moreover, NHP CD8⁺ T cell subsets, with the exception of T_(N), areexpanded in the presence of human IL-15. Anti-CD3/CD2/CD28 beads areused at a 1:2 bead-to-cell ratio to ensure optimal stimulation. IL-7 andIL-15 are both used at 25 ng/mL. However, antibody and cytokineconcentrations are determined according to the experimental need.Optionally, cells are collected and stained for surface antigens asdescribed above.

Helpful hints are described in Table 2.

TABLE 2 Situation Possible reason Solution Cell clumps after thawExcessive cell death Include DNAse in thawing medium. If aggregates arestill seen, sample is filtered over a 70 μm strainer Compbeadsaggregates Sonication was not effective Sonication time is increased to10 min Poor CCR7 staining Presence of NaN₃ in the buffer Buffer withoutNaN₃ is used Staining performed at RT Cells are stained for CCR7 at 37°C. for 20 minutes in a separate step Antibody aggregates are 2 minutesantibody mix Antibody mix is spun for 5-10 minutes seen after stainingcentrifugation was not effective before use Low purity of sorted Poorseparation of antigen Antibody concentration is increased in subsetsexpression due to very high the mix initial cell number Poor separationof antigens Panel is tested carefully before use. due to titrationissues, FMO controls are checked to determine fluorochrome spreading,etc. if compensation is correct and the presence of spreading errors.CFSE dilution is not CFSE aliquot has expired or A new batch of CFSE isused and observed despite has been freezed/thawed tested on fresh PBMCstimulated with increase in cell number in multiple timesanti-CD3/CD2/CD28 beads for 4 days culture before use Sorted cells donot Expired anti-CD3/CD2/CD28 A new batch of anti-CD3/2/38 beads orexpand after stimulation beads or cytokines cytokines is used Cytokineconcentration is too Cytokines are titrated to optimize lowconcentration

The panels indicated here provide the correct identification of humanand NHP primate CD4⁺ and CD8⁺ T_(SCM) cells (FIGS. 11A-11Z). Naïve-likecells, which include both true naïve cells as well as T_(SCM) cells, areidentified using at least three markers and are defined here asCD45RO-CCR7⁺ CD62L⁺ in humans and as CD45RA⁺ CCR7⁺ CD28+ in rhesusmacaques (Table 1, Panel #1 and Panel #3, respectively). In humans, ifusing cryopreserved cells, CD62L is replaced by a different marker (e.g.CD27, Table 1, Panel #2). Within naïve-like cells, a subset expressingCD95, the T_(SCM) population, is identified (FIGS. 11A-11V). Adding anMHC class I tetramer allows the identification of antigen-specificT_(SCM) cells by using the same gating strategy.

The expected frequency of T_(SCM) cells is approximately 2-4% of thetotal CD4⁺ and CD8⁺ T cell populations and does not change appreciablywith the age of the donor. The expected yield after sorting is 1 CD4⁺T_(SCM) cell per every 250 PBMC and 1 CD8⁺ T_(SCM) cell per every500-1,000 PBMC.

For human PBMC, improved identification of the T_(SCM) population isachieved by including CD58 or CD122 in the staining panel, as thesemarkers are differentially expressed in T_(SCM) vs. T_(N) (FIGS.13A-13F). In rhesus macaques, adding CXCR3 ensures better separation ofthe CD8⁺ T_(SCM) subset, as virtually all CD8⁺ T_(SCM) cells are alsoCXCR3⁺ (FIGS. 11E-11V).

Bulk T_(SCM) cells, as well as other subsets, are sorted by flowcytometry at high purity for subsequent genetic analysis, in vitroexpansion and genetic manipulation. Indeed, stimulation withanti-CD3/CD2/CD28 antibody-coated beads or homeostatic cytokines inducescell cycle entry, thus allowing the transduction with retroviral vectors(Cavalieri et al., Blood, 102: 497-505 (2003)). Genetically-modifiedcells are then be utilized for adoptive transfer experiments.

Effective T_(SCM) expansion in vitro is achieved by stimulating withanti-CD3/CD2/CD28 antibody-coated beads (human), plate-bound anti-CD3and soluble anti-CD28 (NHP) or the homeostatic cytokines IL-7 and IL-15.In vitro, CD4⁺ T cells are preferentially expanded by IL-7 while CD8+ Tcells respond to both IL-7 and IL-15. A differential response of humanT_(N) and memory cells is seen with these stimuli, as depicted in FIGS.14A-14L. Thus, expansion conditions are adjusted before proceeding withthe experiment. A combination of both IL-7 and IL-15 is used to maximizeT cell stimulation (Cavalieri et al., Blood, 102: 497-505 (2003)).Stimulation through CD3, CD2 and CD28 expands cells much moreefficiently than homeostatic cytokines but also causes a drastic changein the cell phenotype, including downregulation of CD45RA, CCR7 andCD62L with progressive proliferation as measured by CFSE dilution. Thus,the appropriate stimuli are chosen depending on the application.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of producing an isolated T memory stem cell population, the method comprising (a) isolating lymphocytes from a mammal; and (b) sorting the lymphocytes using flow cytometry into a population comprising a phenotype comprising (i) CD95+ and/or CXCR3+; and (ii) CD45RA+, CCR7+, and CD28+, to produce an isolated T memory stem cell population.
 2. The method of claim 1, further comprising expanding the numbers of T memory stem cells in vitro.
 3. The method of claim 1, further comprising transducing the isolated T memory stem cells with a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
 4. The method of claim 3, wherein the CAR or TCR has antigenic specificity for a cancer antigen or a viral antigen. 